Benzopyran nitrile near-infrared photosensitizer as well as preparation method and application thereof

Abstract

The invention belongs to the field of tumor photodynamic therapy application, discloses a near-infrared photosensitizer as well as a preparation method and application thereof, and particularly relates to a benzopyran nitrile compound shown as a general formula (I). The compound has a near-infrared wavelength luminescence property, can efficiently generate active oxygen to kill tumor cells under an illumination condition, remarkably reduces toxic and side effects, and has good application potential in the field of imaging-induced photodynamic therapy.

Description

Technical Field

[0001] The present invention belongs to the field of tumor photodynamic therapy applications, and specifically relates to a photosensitizer with near-infrared emission properties, a preparation method, and applications thereof. Background Art

[0002] Malignant tumors rank first among the three leading causes of death in humans and are a common threat to human health. Fluorescence imaging-guided precision treatment is crucial. Photodynamic therapy (PDT) is a therapeutic approach in which photosensitizers, under the influence of light, emit fluorescence and generate reactive oxygen species to kill tumor cells. This treatment approach, with its significant advantages such as spatiotemporal controllability, non-invasiveness, and negligible drug resistance, is attracting increasing attention in the application of PDT to solid tumors.

[0003] Near-infrared fluorescent probes have an emission wavelength of up to 700nm, which can avoid the interference of spontaneous fluorescence of samples. At the same time, they have strong penetration ability and reduce tissue damage, which has high efficiency for biological imaging. Designing and developing high-efficiency photosensitizers with near-infrared emission properties for imaging-guided photodynamic therapy can effectively achieve precise treatment of deep tumor tissues and avoid damage to normal tissues, reducing toxic side effects. The present invention aims to provide a new type of benzopyranonitrile photosensitizer that exhibits obvious near-infrared fluorescence emission characteristics and aggregation-induced luminescence properties, as well as strong reactive oxygen species generation capabilities, and apply them to the fields of tumor imaging and photodynamic therapy. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a novel near-infrared benzopyranonitrile photosensitizer. After structural targeting, it can emit strong near-infrared fluorescence under the action of a specific light source and can effectively produce reactive oxygen species, thereby overcoming the problems of weak tissue penetration and strong background interference of traditional photosensitizers.

[0005] Another object of the present invention is to disclose a method for preparing the photosensitive material, which method is highly operable and efficient.

[0006] Another object of the present invention is to clarify the photophysical properties and reactive oxygen generation performance of the above-mentioned photosensitizer and apply them to the fields of cell imaging and photodynamic therapy.

[0007] Technical solution: The benzopyranonitrile photosensitizer with near-infrared emission characteristics of the present invention comprises a compound having a structure as shown in general formula (I):

[0008]

[0009] in,

[0010] R1 is selected from one of the following structural fragments:

[0011]

[0012] R2 is selected from one of the following structural fragments:

[0013]

[0014] The preparation method of the compound of general formula (I) comprises the following steps:

[0015] 2-Hydroxy-5-bromoacetophenone is used as the starting material, and 2-hydroxy-5-bromoacetophenone is reacted with ethyl acetate to obtain a bromobenzopyrone intermediate 1, which is then condensed with malononitrile to obtain a bromobenzopyrancarbonitrile intermediate 2. This is then condensed with different aromatic aldehyde compounds via a Knoevenagel reaction to obtain an intermediate 3, which then undergoes a Heck coupling reaction with a vinyl-substituted aromatic group, and finally reacts with iodomethane and undergoes ion exchange to obtain a compound of formula (I).

[0016]

[0017] Reaction conditions: (a) sodium hydride, ethyl acetate, THF, 65°C, 3h; (b) concentrated hydrochloric acid, methanol, rt, overnight; (c) malononitrile, acetic anhydride, 140°C, 12h; (d) aromatic groups substituted with different aldehydes, piperidine, ethanol, 80°C, 4h; (e) aromatic groups substituted with vinyl groups, palladium acetate, tri(o-methylphenyl)phosphine, triethylamine, DMF, 95°C, 4h; (f) aromatic groups substituted with vinyl groups, tetrabutylammonium bromide, potassium acetate, palladium acetate, DMF, 80°C, 2h; (g) iodomethane, acetonitrile, 80°C, 12h; (h) saturated aqueous potassium hexafluorophosphate solution, rt, 4h.

[0018] Preferably, the compound is selected from the following compounds:

[0019]

[0020] The preferred method for preparing the compound comprises the following steps:

[0021] (1) Synthesis route of probe molecules PS1-PS4:

[0022] 2-Hydroxy-5-bromoacetophenone was used as the starting material, and 2-hydroxy-5-bromoacetophenone was reacted with ethyl acetate to obtain bromobenzopyrone intermediate 1, which was condensed with malononitrile to obtain bromobenzopyranonitrile intermediate 2. Intermediate 3 was condensed with different aromatic aldehyde compounds through Knoevenagel reaction, and then intermediate 4 was obtained by Heck coupling reaction with 4-vinylpyridine. Finally, intermediate 4 was reacted with iodomethane (5) and subjected to ion exchange in saturated potassium hexafluorophosphate aqueous solution to obtain probe molecules PS1-PS4.

[0023]

[0024] Reaction conditions: (a) sodium hydride, ethyl acetate, THF, 65°C, 3h; (b) concentrated hydrochloric acid, methanol, rt, overnight; (c) malononitrile, acetic anhydride, 140°C, 12h; (d) aromatic groups substituted with different aldehydes, piperidine, ethanol, 80°C, 4h; (e) 4-vinylpyridine, palladium acetate, tri(o-methylphenyl)phosphine, triethylamine, DMF, 95°C, 4h; (f) 4-vinylpyridine, tetrabutylammonium bromide, potassium acetate, palladium acetate, DMF, 80°C, 2h; (g) iodomethane, acetonitrile, 80°C, 12h; (h) saturated aqueous potassium hexafluorophosphate solution, rt, 4h.

[0025] (2) Synthesis route of probe molecule PS5

[0026] 2-Hydroxy-5-bromoacetophenone was used as the starting material, and 2-hydroxy-5-bromoacetophenone was reacted with ethyl acetate to obtain bromobenzopyrone intermediate 1, which was condensed with malononitrile to obtain bromobenzopyrancarbonitrile intermediate 2. The intermediate 3b was condensed with 4-dimethylaminobenzaldehyde via Knoevenagel reaction, and then reacted with 4-vinylquinoline to obtain intermediate 6 via Heck coupling reaction. Finally, the intermediate 6 was reacted with iodomethane (7) and subjected to ion exchange in saturated potassium hexafluorophosphate aqueous solution to obtain the probe molecule PS5.

[0027]

[0028] Reaction conditions: (a) sodium hydride, ethyl acetate, THF, 65°C, 3h; (b) concentrated hydrochloric acid, methanol, rt, overnight; (c) malononitrile, acetic anhydride, 140°C, 12h; (d) 4-diphenylaminobenzaldehyde, piperidine, ethanol, 80°C, 4h; (e) 4-vinylpyridine, palladium acetate, tri(o-methylphenyl)phosphine, triethylamine, DMF, 95°C, 4h; (f) iodomethane, acetonitrile, 80°C, 12h; (g) saturated aqueous potassium hexafluorophosphate solution, rt, 4h.

[0029] The compound can emit fluorescence in a wavelength range greater than 700 nm.

[0030] The compound can be used as a photosensitizer to effectively generate active oxygen to kill tumor cells.

[0031] The compound is used in the fields of organelle and tumor imaging and tumor photodynamic therapy.

[0032] The present invention has the following advantages and beneficial effects:

[0033] 1. The benzopyranonitrile photosensitizer provided by the present invention utilizes the non-planar structure of triphenylamine to effectively inhibit intermolecular π-π interactions, exhibit aggregation-induced emission effect, and enhance the ability to generate reactive oxygen species.

[0034] 2. The benzopyranonitrile photosensitizer provided by the present invention utilizes a strong D-π-A system structure, which can emit near-infrared fluorescence greater than 700nm under excitation of longer wavelengths, reducing tissue interference and enhancing tissue penetration.

[0035] 3. The raw materials of the benzopyranonitrile photosensitizer provided by the present invention are easily available and simple to synthesize. There are no similar reports and the benzopyranonitrile photosensitizer has strong commercial value.

[0036] 4. The benzopyranonitrile photosensitizer provided by the present invention has been successfully applied to the targeted positioning and imaging of organelles in living cells, providing a new tool for the field of imaging-guided photodynamic therapy of tumors and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings are used to provide further explanation of the present invention and constitute a part of the specification. Together with the following detailed description, they are used to explain the present invention but do not constitute a limitation of the present invention.

[0038] Figure 1 Emission spectrum of probe PS1

[0039] Figure 2 Emission spectrum of probe PS2

[0040] Figure 3 Emission spectrum of probe PS3

[0041] Figure 4 Emission spectrum of probe PS4

[0042] Figure 5 Emission spectrum of probe PS5

[0043] Figure 6 Photosensitive activity of probe PS1 under light conditions

[0044] Figure 7 Photosensitivity of probe PS2 under light conditions

[0045] Figure 8 Photosensitive activity of probe PS4 under light conditions

[0046] Figure 9 Photosensitivity of probe PS5 under light conditions

[0047] Figure 10 Cytotoxicity of probe PS2 under dark and light conditions

[0048] Figure 11Fluorescence imaging of PS2 probe in living cells Specific implementation methods

[0049] (The embodiments are intended to illustrate the present invention, rather than to limit the present invention)

[0050] The novel features of the present invention are particularly set forth in the claims. Exemplary embodiments utilizing the principles of the present invention are set forth below. The features and advantages of the present invention may be better understood by reference to the following.

[0051] Although preferred embodiments of the present application are described herein, these embodiments are provided as examples only. It should be understood that the variations of the embodiments of the present application described herein may also be used to implement the technical solutions of the present application. Those of ordinary skill in the art will appreciate that various variations, changes, and substitutions may occur without departing from the scope of the present application. It should be understood that the scope of protection of each aspect of the present application is determined by the claims, and the methods and structures within the scope of these claims and their equivalents are within the scope encompassed by the claims of the present application.

[0052] The preparation of some of the compounds is as follows:

[0053] 1 H-NMR nuclear magnetic resonance was measured by a Bruker AV400 (400 Hz) nuclear magnetic resonance instrument (TMS was used as the internal standard), and mass spectra were measured by a Shimadzu GC / MS-QP2010 mass spectrometer (EI-MS) and an Agilent 100LC-MDS-Trans / SL mass spectrometer (EI-MS), respectively.

[0054] Column chromatography silica gel was 300-400 mesh (Qingdao Ocean Chemical Plant), and the eluent was petroleum ether-ethyl acetate or dichloromethane-methanol. Thin-layer chromatography (TLC) used GF254 thin-layer chromatography plates (Yantai Huayang New Materials Technology Co., Ltd.). The TLC developing system was petroleum ether-ethyl acetate or dichloromethane-methanol. TLC was visualized using a ZF7 triple-UV analyzer (Henan Gongyi Yuhua Instrument Co., Ltd.).

[0055]

[0056] Example 1: Synthesis of Intermediate 1

[0057] Sodium hydride (2.23 g, 55.8 mmol) was suspended in THF, stirred at room temperature for 2 minutes under argon, and 2-hydroxy-5-bromoacetophenone (3 g, 14 mmol) and ethyl acetate (3.4 mL, 34.9 mmol) were dissolved in THF and slowly added to the reaction system. The mixture was heated to 65°C, refluxed under argon for 3 hours, and the reaction was complete as monitored by TLC. The reaction solution was poured into ice water, and the pH was adjusted to 5-6 with 2N hydrochloric acid. The mixture was extracted with ethyl acetate (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a concentrated crude product. The crude product was dissolved in anhydrous ethanol, and 2 mL of concentrated hydrochloric acid was added dropwise. The reaction was stirred at room temperature overnight. The reaction was complete as monitored by TLC. The solvent was removed under reduced pressure and purified by column chromatography to obtain 3.2 g of a white solid in a yield of 96.0%.

[0058] Example 2: Synthesis of Intermediate 2

[0059] Intermediate 1 (1.5 g, 6.3 mmol) and malononitrile (1.25 g, 18.8 mmol) were dissolved in acetic anhydride and refluxed at 140°C under argon for 12 h. After completion of the reaction as monitored by TLC, the majority of the solvent was removed under reduced pressure. 20 mL of deionized water was added, and the mixture was stirred and refluxed at 90°C for 0.5 h under argon. The reaction solution was extracted with dichloromethane (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent removed under reduced pressure. Purification by column chromatography afforded 680 mg of a light yellow solid in a 37.8% yield. 1 H NMR (400MHz, Chloroform-d) δ9.05(d,J=2.2Hz,1H),7.80(dd,J=8.9,2.2Hz,1H),7.34(d,J=8.9Hz,1H),6.74–6.67(m,1H),2.43(s,3H).

[0060] Example 3: Synthesis of Intermediate 3a

[0061] Intermediate 2 (50 mg, 0.17 mmol) and p-dimethylaminobenzaldehyde (52 mg, 0.35 mmol) were dissolved in anhydrous ethanol under argon atmosphere. The mixture was stirred at reflux at 80°C for 10 min. Piperidine (35 μL, 0.35 mmol) was added dropwise. The mixture was refluxed at 80°C under argon atmosphere for 4 h. The reaction was monitored for completion by TLC. The mixture was cooled, filtered, washed, and dried to obtain 52 mg of a dark red solid (yield 71.7%). 1H NMR(400MHz,Chloroform-d)δ9.04(d,J=2.2Hz,1H),7.79(dd,J=8.9,2.2Hz,1H),7.56(d,J=15.8Hz,1H),7.50( d,J=9.0Hz,2H),7.42(d,J=8.9Hz,1H),6.83(d,J=8.4Hz,2H),6.79(s,1H),6.59(d,J=15.8Hz,1H),3.09(s,6H).

[0062] Example 4: Synthesis of Intermediate 4a

[0063] Intermediate 3a (20 mg, 0.048 mmol), palladium acetate (0.8 mg, 0.0048 mmol) and tri(o-methylphenyl)phosphine were dissolved in a mixed solvent of triethylamine and DMF (V TEA :V DMF =2:1) ​​under argon protection, stirred at 30°C for 25 min, added 4-vinylpyridine (42 μL, 0.38 mmol), protected by argon, refluxed at 95°C for 4 h, and the reaction was completed after monitoring by TLC. The solvent was removed under reduced pressure and purified by column chromatography to obtain 8 mg of an orange solid in a yield of 38%. 1 H NMR(500MHz,Chloroform-d)δ9.08(s,1H),8.61(d,J=5.1Hz,2H),7.89(d,J=7.2Hz,1H),7.61–7.54(m,2H),7.49(d,J=8.7Hz,2H),7.41( d,J=5.1Hz,2H),7.34(d,J=16.3Hz,1H),7.10(d,J=16.2Hz,1H),6.80(s,1H),6.72(d,J=8.8Hz,2H),6.59(d,J=15.7Hz,1H),3.08(s,6H).

[0064] Example 5: Synthesis of Intermediate 5a

[0065] Intermediate 4a (8 mg, 0.018 mmol) was dissolved in ultra-dry THF, and iodomethane (20 μL, 0.33 mmol) was added. Under argon protection, the mixture was refluxed at 80°C for 12 h. The reaction was completed after monitoring by TLC. The solvent was removed under reduced pressure and purified by column chromatography to obtain 6 mg of a red solid in a yield of 56.6%.

[0066] Example 6: Synthesis of PS1

[0067] Intermediate 5a (5 mg, 0.008 mmol) was dissolved in THF, and saturated aqueous potassium hexafluorophosphate solution was added. The mixture was stirred at room temperature for 4 h, filtered, washed, and dried to obtain 5 mg of a red solid with a yield of 97.1%. 1 H NMR (600MHz, DMSO-d6) δ8.92(d,J=1.9Hz,1H),8.90(d,J=6.5Hz,2H),8.33(dd,J=8.9,2.0Hz,1 H),8.30(d,J=6.5Hz,2H),8.05(d,J=16.3Hz,1H),7.93(d,J=8.8Hz,1H),7.71(d,J=15.8Hz,1H ),7.64(d,J=8.5Hz,2H),7.54(d,J=16.3Hz,1H),7.39(t,J=7.9Hz,4H),7.34(d,J=15.9Hz,1H) ,7.18(t,J=7.4Hz,2H),7.14(d,J=7.8Hz,4H),7.01(s,1H),6.92(d,J=8.4Hz,2H),4.28(s,3H).

[0068]

[0069] Example 7: Synthesis of Intermediate 3b

[0070] Intermediate 2 (50 mg, 0.17 mmol) and 4-diphenylaminobenzaldehyde (95 mg, 0.35 mmol) were dissolved in anhydrous ethanol under argon atmosphere. The mixture was stirred at reflux at 80°C for 10 min. Piperidine (35 μL, 0.35 mmol) was added dropwise to the reaction mixture. The mixture was refluxed at 80°C for 4 h under argon atmosphere. The reaction was monitored for completion by TLC. The mixture was cooled, filtered, washed, and dried to obtain 76 mg of a dark red solid (yield 80.9%). 1 HNMR(500MHz,Chloroform-d)δ9.05(d,J=2.2Hz,1H),7.80(dd,J=9.0,2.2Hz,1H),7.54(d,J=15.8Hz,1H),7.42(t,J=8 .4Hz,3H),7.32(dd,J=8.4,7.2Hz,4H),7.18–7.12(m,6H),7.03(d,J=8.6Hz,2H),6.81(s,1H),6.63(d,J=15.8Hz,1H).

[0071] Example 8: Synthesis of Intermediate 4b

[0072] Intermediate 3b (30 mg, 0.055 mmol), palladium acetate (1.0 mg, 0.0055 mmol) and tri(o-methylphenyl)phosphine (3.5 mg, 0.011 mmol) were dissolved in a mixed solvent of triethylamine and DMF (V TEA :V DMF =2:1) ​​under argon protection, stirred at 30°C for 25 min, added 4-vinylpyridine (48 μL, 0.44 mmol) to the reaction solution, under argon protection, refluxed at 95°C for 4 h. The reaction was completed after monitoring by TLC, and the solvent was removed under reduced pressure. The mixture was purified by column chromatography to obtain 9 mg of an orange solid in a yield of 28.8%. 1 H NMR(400MHz,Chloroform-d)δ9.06(d,J=2.0Hz,1H),8.65–8.62(m,2H),7.90(dd,J=8.8,2.0Hz,1H),7.58(s, 1H),7.55(d,J=7.4Hz,1H),7.45(d,J=1.6Hz,1H),7.43(q,J=1.8Hz,2H),7.41(d,J=2.1Hz,1H),7.35(d,J=4. 7Hz,1H),7.33(d,J=1.2Hz,2H),7.31(d,J=2.1Hz,1H),7.31(s,1H),7.17(d,J=1.3Hz,2H),7.15(dd,J=2.0,0 .9Hz,2H),7.14(d,J=1.5Hz,1H),7.12–7.11(m,1H),7.05–7.02(m,2H),6.80(s,1H),6.63(d,J=15.8Hz,1H).

[0073] Example 9: Synthesis of Intermediate 5b

[0074] Intermediate 4b (9 mg, 0.016 mmol) was dissolved in acetonitrile, and iodomethane (20 μL, 0.33 mmol) was added. Under argon protection, the mixture was refluxed at 80°C for 12 h. The reaction was completed after monitoring by TLC. The solvent was removed under reduced pressure and purified by column chromatography to obtain 9 mg of a red solid in a yield of 97.8%.

[0075] Example 10: Synthesis of PS2

[0076] Intermediate 5b (7 mg, 0.012 mmol) was dissolved in THF, and saturated aqueous potassium hexafluorophosphate solution was added. The mixture was stirred at room temperature for 4 h, filtered, washed, and dried to obtain 6 mg of a red solid with a yield of 69.0%. 1H NMR (600MHz, DMSO-d6) δ8.92(d,J=1.9Hz,1H),8.90(d,J=6.5Hz,2H),8.33(dd,J=8.9,2.0Hz,1 H),8.30(d,J=6.5Hz,2H),8.05(d,J=16.3Hz,1H),7.93(d,J=8.8Hz,1H),7.71(d,J=15.8Hz,1H ),7.64(d,J=8.5Hz,2H),7.54(d,J=16.3Hz,1H),7.39(t,J=7.9Hz,4H),7.34(d,J=15.9Hz,1H) ,7.18(t,J=7.4Hz,2H),7.14(d,J=7.8Hz,4H),7.01(s,1H),6.92(d,J=8.4Hz,2H),4.28(s,3H).

[0077]

[0078] Example 11: Synthesis of Intermediate 3c

[0079] Intermediate 2 (21 mg, 0.07 mmol) and 4-(9H-carbazol-9-yl)benzaldehyde (40 mg, 0.15 mmol) were dissolved in anhydrous ethanol under argon atmosphere. The mixture was stirred at reflux at 80°C for 10 min. Piperidine (15 μL, 0.15 mmol) was added dropwise to the reaction mixture. The mixture was refluxed at 80°C for 4 h under argon atmosphere. The reaction was monitored for completion by TLC. The mixture was cooled, filtered, washed, and dried to obtain 26 mg of a dark red solid (yield 65.8%). 1 H NMR (400MHz, DMSO-d6) δ8.88(d,J=2.2Hz,1H),8.26(d,J=7.7Hz,2H),8.12(dd,J=9.0,2.3Hz,1H),8.08–8.03(m,2H),7.91(d,J=16.0Hz, 1H),7.82(d,J=8.9Hz,1H),7.78(d,J=8.4Hz,2H),7.70–7.64(m,1H),7.52–7.43(m,4H),7.32(ddd,J=7.9,6.7,1.4Hz,2H),7.13(s,1H).

[0080] Example 12: Synthesis of Intermediate 4c

[0081] Intermediate 3c (30 mg, 0.056 mmol), palladium acetate (1.0 mg, 0.006 mmol) and tri(o-methylphenyl)phosphine (3.4 mg, 0.01 mmol) were dissolved in a mixed solvent of triethylamine and DMF (V TEA :V DMF=2:1) ​​under argon protection, stirred at 30°C for 25 min, added 4-vinylpyridine (30 μL, 0.28 mmol) to the reaction solution, under argon protection, refluxed at 95°C for 4 h, the reaction was completed after monitoring by TLC, the solvent was removed under reduced pressure, and purified by column chromatography to obtain 13 mg of an orange solid in a yield of 41.5%. 1 H NMR (400MHz, DMSO-d6) δ8.91(d,J=2.0Hz,1H),8.61–8.56(m,2H),8.27(dd,J=7.1,1.7Hz,3H),8.07(d,J=8.5Hz,2H),7.92(d,J=3.6Hz,1H),7.89(d ,J=3.5Hz,1H),7.78(d,J=8.5Hz,2H),7.67(d,J=16.1Hz,1H),7.62–7.58 (m,3H),7.52–7.45(m,4H),7.32(td,J=7.3,6.8,1.4Hz,3H),7.14(s,1H).

[0082] Example 13: Synthesis of Intermediate 5c

[0083] Intermediate 4c (13 mg, 0.023 mmol) was dissolved in acetonitrile, and iodomethane (30 μL, 0.48 mmol) was added. Under argon protection, the reaction was refluxed at 80°C for 12 h. The reaction was completed after monitoring by TLC. The solvent was removed under reduced pressure and purified by column chromatography to obtain 10 mg of an orange solid in a yield of 61.3%.

[0084] Example 14: Synthesis of PS3

[0085] Intermediate 5c (5 mg, 0.007 mmol) was dissolved in THF, and saturated aqueous potassium hexafluorophosphate solution was added. The mixture was stirred at room temperature for 4 h, filtered, washed, and dried to obtain 4 mg of an orange solid with a yield of 78.4%. 1 H NMR(600MHz,DMSO-d6)δ8.93–8.85(m,2H),8.35(d,J=8.8Hz,1H),8.29–8.1 5(m,5H),8.06–7.99(m,2H),7.95(d,J=8.7Hz,1H),7.88(dd,J=16.5,7.7Hz ,1H),7.75(d,J=8.0Hz,2H),7.66(d,J=16.2Hz,1H),7.56–7.51(m,1H),7.4 6(dt,J=14.9,7.9Hz,4H),7.30(t,J=7.3Hz,3H),7.12(s,1H),4.26(s,3H).

[0086]

[0087] Example 15: Synthesis of Intermediate 3d

[0088] Intermediate 2 (50 mg, 0.17 mmol) and 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde (93 mg, 0.26 mmol) were dissolved in anhydrous ethanol under argon atmosphere. The mixture was stirred at reflux at 80°C for 10 min. Piperidine (35 μL, 0.35 mmol) was added dropwise to the reaction mixture. The mixture was refluxed at 80°C for 4 h under argon atmosphere. The reaction was monitored for completion by TLC. The mixture was cooled, filtered, washed, and dried to obtain 95 mg of a dark red solid (yield 87.2%). 1 H NMR(400MHz,Chloroform-d)δ9.04(d,J=2.2Hz,1H),7.80(dd,J=9.0,2.2Hz,1H),7.68(d,J=15.5Hz,1H),7.50–7.45(m,2H),7.41(d ,J=8.9Hz,1H),7.32–7.27(m,5H),7.22(d,J=3.9Hz,1H),7.16–7.12(m,4H),7.11–7.05(m,4H),6.81(s,1H),6.52(d,J=15.5Hz,1H).

[0089] Example 16: Synthesis of Intermediate 4d

[0090] Intermediate 3d (200 mg, 0.32 mmol), 4-vinylpyridine (42 μL, 0.38 mmol), tetrabutylammonium bromide (155 mg, 0.48 mmol), palladium acetate (16 mg, 0.096 mmol) and potassium acetate (50 mg, 0.51 mmol) were dissolved in DMF. Under argon protection, the reaction was carried out in a sealed tube at 80° C. for 2 h. The reaction was monitored for completion by TLC. The solvent was removed under reduced pressure and purified by column chromatography to give 90 mg of a dark red solid in a yield of 43.3%. 1 H NMR(400MHz,Chloroform-d)δ9.08(d,J=2.0Hz,1H),8.61(s,2H),7.90(dd,J=8.8 ,2.0Hz,1H),7.70(d,J=15.5Hz,1H),7.56(d,J=8.7Hz,1H),7.50–7.46(m,2H),7. 44(d,J=5.2Hz,2H),7.32(d,J=4.9Hz,2H),7.31–7.27(m,5H),7.22(d,J=3.9Hz,1 H),7.16–7.12(m,5H),7.08(d,J=3.4Hz,3H),6.82(s,1H),6.53(d,J=15.5Hz,1H).

[0091] Example 17: Synthesis of Intermediate 5d

[0092] Intermediate 4d (86 mg, 0.13 mmol) was dissolved in ultra-dry acetonitrile, and iodomethane (83 μL, 1.33 mmol) was added. Under argon protection, the mixture was refluxed at 80°C for 12 h. The reaction was completed after monitoring by TLC. The solvent was removed under reduced pressure and purified by column chromatography to obtain 80 mg of a dark red solid in a yield of 76.3%.

[0093] Example 18: Synthesis of PS4

[0094] Intermediate 5d (80 mg, 0.1 mmol) was dissolved in acetonitrile, and saturated aqueous potassium hexafluorophosphate solution was added. The mixture was stirred at room temperature for 4 h, filtered, washed, and dried to obtain 80 mg of a dark red solid with a yield of 97.8%. 1 H NMR (600MHz, DMSO-d6) δ8.84(d,J=12.3Hz,3H),8.28(d,J=8.6Hz,1H),8.23(d,J =5.9Hz,2H),7.97(d,J=16.1Hz,2H),7.86–7.81(m,2H),7.56(d,J=8.3Hz,2H),7. 50(d,J=3.5Hz,1H),7.48–7.43(m,2H),7.35(t,J=7.8Hz,4H),7.12(t,J=7.4Hz,2 H),7.07(d,J=7.8Hz,4H),7.04–7.01(m,1H),6.95(d,J=8.3Hz,2H),4.23(s,3H).

[0095]

[0096] Example 19: Synthesis of Intermediate 6

[0097] Intermediate 3b (30 mg, 0.055 mmol), 4-vinylquinoline (9.4 mg, 0.061 mmol), tetrabutylammonium bromide (26.7 mg, 0.083 mmol), palladium acetate (0.46 mg, 0.003 mmol) and potassium acetate (8.7 mg, 0.088 mmol) were dissolved in DMF. The reaction was carried out in a sealed tube at 80°C under argon protection for 2 h. The reaction was monitored for completion by TLC. The solvent was removed under reduced pressure and the product was purified by column chromatography to give 16 mg of a dark red solid in a yield of 47.1%. 1H NMR(400MHz,Chloroform-d)δ9.26(d,J=2.0Hz,1H),8.93(d,J=4.6Hz,1H),8.27(d,J=8.3Hz ,1H),8.15(d,J=8.4Hz,1H),8.00–7.96(m,1H),7.96–7.93(m,1H),7.76(ddd,J=8.4,6.8,1.4 Hz,1H),7.66–7.60(m,3H),7.58(d,J=11.4Hz,1H),7.45(s,1H),7.42(d,J=3.5Hz,2H),7.35 –7.31(m,4H),7.19–7.13(m,6H),7.04(d,J=8.7Hz,2H),6.86(s,1H),6.67(d,J=15.8Hz,1H).

[0098] Example 20: Synthesis of Intermediate 7

[0099] Intermediate 6 (15 mg, 0.024 mmol) was dissolved in acetonitrile, and iodomethane (30 μL, 0.48 mmol) was added. Under argon protection, the reaction was refluxed at 80°C for 12 h. The reaction was completed after monitoring by TLC. The solvent was removed under reduced pressure and purified by column chromatography to obtain 8 mg of a dark red solid in a yield of 43.5%.

[0100] Example 21: Synthesis of PS5

[0101] Intermediate 7 (5 mg, 0.0066 mmol) was dissolved in THF, and saturated aqueous potassium hexafluorophosphate solution was added. The mixture was stirred at room temperature for 4 h, filtered, washed, and dried to obtain 4 mg of an orange solid with a yield of 78.4%. 1 H NMR (400MHz, DMSO-d6) δ9.40(d,J=6.6Hz,1H),9.02(s,1H),8.96(d,J=8.5Hz,1H),8.64(d,J=9.7H z,1H),8.60(d,J=6.4Hz,1H),8.46(d,J=8.8Hz,1H),8.29–8.23(m,1H),8.14(d,J=15.9Hz,1H),8. 08–8.03(m,1H),7.94(d,J=8.8Hz,1H),7.70(d,J=15.7Hz,1H),7.62(d,J=8.7Hz,2H),7.39–7.32( m,5H),7.15(d,J=7.6Hz,2H),7.13–7.08(m,5H),7.00(s,1H),6.89(d,J=8.6Hz,2H),4.55(s,3H).

[0102] Experimental Example 1

[0103] 1. Absorption and emission spectra of probe molecules

[0104] (1) Prepare a DMSO stock solution (10 mM) of the compound probe molecule in the example, and then dilute it with DMSO to a probe molecule test solution with a final concentration of 10 μM.

[0105] (2) Add the above probe molecule test solution into a 96-well plate, set up 2 replicate wells for each sample, 200 μL per well, and then use Tecan Spark TM The absorption spectra of the compounds were tested using a 10M Multimode Microplate Reader, and the maximum absorption wavelength and maximum emission wavelength of each probe molecule were recorded, as shown in Table 1.

[0106] (3) The DMSO stock solution (10 mM) was diluted with DMSO or different ratios of toluene / DMSO solvent system to a final concentration of 10 μM, wherein the ratio of toluene was 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% in sequence. Based on the maximum absorption wavelength obtained above, the emission spectrum of the probe molecule in the above system was excited by the maximum absorption wavelength, such as Figures 1 to 5 shown.

[0107] 2. Determination of the molar extinction coefficient of the probe molecule

[0108] The above probe molecule stock solution was diluted with H2O to a probe molecule test solution with a final concentration of 10 μM. The absorbance of the probe molecule was tested on a microplate reader using a quartz cuvette, and then the molar extinction coefficient was calculated, as shown in Table 1.

[0109] 3. Determination of the Reactive Oxygen Species Generating Ability of Probe Molecules

[0110] ABDA (100 μM) was used as the active oxygen indicator, and photosensitizer (10 μM) was added to test the absorption curve of ABDA after different illumination times. Figures 6 to 9 shown.

[0111] Table 1 Photophysical properties of probe molecules

[0112]

[0113] Experimental Example 2

[0114] Photosensitizer cytotoxicity test: HeLa cells were seeded in 96-well plates (5×10 3 / well) and cultured in a CO2 incubator (37°C, 5% CO2) with DMEM medium containing 10% fetal bovine serum for 24 hours. Subsequently, photosensitizer PS2 was added at final concentrations of 20μM, 10μM, 5μM, and 1μM, respectively, and incubated for 4 hours. Illuminate for 15 minutes and continue to culture for 24 hours. 20μL / well MTT (5mg / mL) was added and incubated for another 4 hours. The medium was removed, and 150μL DMSO was added to each well. The OD value at 492nm was read and the cell survival rate was calculated, as shown in Table 2 and Figure 10 The results showed that the photosensitizer PS2 had concentration-dependent phototoxicity.

[0115] Table 2 Cytotoxicity of photosensitizer PS2

[0116]

[0117] Experimental Example 3

[0118] Live cell imaging experiment: HeLa cells were seeded in a confocal culture dish (2×10 5 / mL), placed in a CO2 incubator (37°C, 5% CO2) and cultured in DMEM medium containing 10% fetal bovine serum for 24 hours. The medium containing the photosensitizer PS2 at a final concentration of 1 μM was added and incubated for 1 hour. The medium was removed, and the cells were washed three times with PBS (pH = 7.4). Images were collected under a Leica confocal microscope, as shown. Figure 11 The results showed that the photosensitizer PS2 can achieve live cell imaging.

Claims

1. A benzopyran nitrile compound represented by formula (I) or a pharmaceutically acceptable salt thereof: in, R1 is selected from one of the following structural fragments: R2 is selected from one of the following structural fragments:

2. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, characterized in that The compound is selected from the following compounds:

3. The method for preparing the compound according to claim 1 or 2, characterized in that: The following steps are involved: (1) Synthesis route of probe molecules PS1-PS4: Using 4-bromo-2-hydroxyacetophenone as the starting material, cyclization is performed to obtain bromobenzopyrone intermediate 1, which is then condensed with malononitrile to obtain bromobenzopyranonitrile intermediate 2, which is then condensed with different aromatic aldehyde compounds via Knoevenagel reaction to obtain intermediate 3, which is then subjected to Heck coupling reaction with 4-vinylpyridine to obtain intermediate 4, which is finally reacted with iodomethane (5) and subjected to ion replacement to obtain probe molecules PS1-PS4; (2) Synthesis route of probe molecule PS5: Starting from 4-bromo-2-hydroxyacetophenone, cyclization was performed to obtain bromobenzopyrone intermediate 1, which was condensed with malononitrile to obtain bromobenzopyrancarbonitrile intermediate 2. This was coupled with 4-diphenylaminobenzaldehyde via Knoevenagel reaction to obtain intermediate 3b, which was then subjected to Heck coupling reaction with 4-vinylquinoline to obtain intermediate 6. Finally, it was reacted with iodomethane (7) and ion exchange to obtain the probe molecule PS5.

4. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of a near-infrared photosensitizer.

5. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of an optical imaging agent.

6. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of drugs for photodynamic therapy of tumors.

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